Manipulation of light spectrum can improve the performance of photosynthetic apparatus of strawberry plants growing under salt and alkalinity stress

Strawberry is one of the plants sensitive to salt and alkalinity stress. Light quality affects plant growth and metabolic activities. However, there is no clear answer in the literature on how light can improve the performance of the photosynthetic apparatus of this species under salt and alkalinity stress. The aim of this work was to investigate the effects of different spectra of supplemental light on strawberry (cv. Camarosa) under salt and alkalinity stress conditions. Light spectra of blue (with peak 460 nm), red (with peak 660 nm), blue/red (1:3), white/yellow (1:1) (400–700 nm) and ambient light were used as control. There were three stress treatments: control (no stress), alkalinity (40 mM NaHCO3), and salinity (80 mM NaCl). Under stress conditions, red and red/blue light had a positive effect on CO2 assimilation. In addition, blue/red light increased intrinsic water use efficiency (WUEi) under both stress conditions. Salinity and alkalinity stress decreased OJIP curves compared to the control treatment. Blue light caused an increase in its in plants under salinity stress, and red and blue/red light caused an increase in its in plants under alkalinity. Both salt and alkalinity stress caused a significant reduction in photosystem II (PSII) performance indices and quantum yield parameters. Adjustment of light spectra, especially red light, increased these parameters. It can be concluded that the adverse effects of salt and alkalinity stress on photosynthesis can be partially alleviated by changing the light spectra.


Introduction
Light is the plant's primary energy supply, which stimulates various responses. It is a major environmental factor that affects the control of plant growth and development [1]. Plant a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 four different light variants on chlorophyll fluorescence and plant gas exchange parameters of strawberry cv. Camarosa under salinity and alkalinity stress. Salinity and alkalinity are stresses that inhibit plant growth, and much research has been done to reduce the effects of these stresses on plants. So, we hypothesize that different wavelengths of light affect plants' response to these stresses by counteracting stress-induced imbalances in the functioning of the photosynthetic apparatus. This type of study is necessary to analyze the response of plants to lighting systems under stress conditions and to use LEDs with different wavelengths as radiation sources for plant research and horticultural production.

Plant material and growth conditions
This experiment was conducted in the Vali-e-Asr University experimental greenhouse in 2020. The rooted strawberry plants (Fragaria × ananassa Duch, cV. Camarosa) were obtained from a nursery in Karaj, Iran. Plants were planted in a pot of 4 liters containing cocopeat and perlite (70: 30). The plants were growing in a greenhouse with a temperature of 25/15±2˚C (day/night), 11/13 h (light/dark) photoperiod, and relative humidity of 50±10%. During the growing period, irrigation of plants was done with Morgan nutrient solution [28] (EC: 1.4 dS m -1 , pH: 6.5) ( Table 1). Plants were treated by five light levels and three stress levels including: control (without stress), salinity (80 mM NaCl), and alkalinity (40 mM NaHCO 3 ). To simulate salinity and alkalinity stresses under natural growth conditions (accumulation of salts during following growth stages) at first, salinity stress was induced by 40 mM NaCl and alkalinity by 20 mM NaHCO 3 . Then after, the concentrations of salt and alkalinity were increased until the effects of stress were observed on plants and the final concentrations reached 80 mM NaCl and 40 mM NaHCO 3 . The plants were stressed for 60 days and given NaCl and NaHCO 3 every 4 days. Each treatment included three pots, and each pot had three plants.

Leaf gas exchange
Photosynthetic parameters include CO 2 assimilation rate (A), stomatal conductance (g s ), transpiration rate (E), sub-stomatal CO 2 concentration (C i ), instantaneous carboxylation efficiency (A/C i ), and intrinsic water-use efficiency (WUE i ) were measured 60 days after planting with portable photosynthesis system (ADC Bio Scientific Ltd, LCi-sd, Hoddesdon U.K.). Measurements were performed on fully grown leaves, between 9:00 AM and 12:00 AM. The air temperature and light intensity in cuvette during measurements were ambient. The CO 2 concentration was 400 ppm.

Experimental design and data analysis
The experiment was a complete randomized design with two factors in three replications in factorial form and three single-plant per pot. Using SAS software to analyze all data (SAS Institute, Cary, NC, USA). All data were statistically analyzed using two-way ANOVA model. When analysis of variance (ANOVA) indicated significant treatment effects, significant mean differences (P<0.05) were calculated by the LSD Multiple Range Test as a post hoc. Once the differences between the means are demonstrated, it is possible to determine which means are different using post hoc range tests and pairwise multiple comparisons. Range tests identify homogeneous subsets of means that do not differ from each other. The chlorophyll fluorescence parameters were calculated using the software "PEA Plus" version 1.12 (Hansatech). Pearson's correlation coefficient was applied to determine the relationships among the parameters studied. The graphs were made using Excel 2013 (Microsoft, Redmont, WA, USA).

Leaf gas exchange analyses
Analysis of ANOVA (Table 4) shows different light spectra, stresses, and, what's most important the interactions between them affect the gas exchange parameters of plants. CO 2 assimilation rate (A) of plants was influenced considerably by salinity and alkalinity stress and different light spectra (Fig 3). CO 2 assimilation rate decreased under stress conditions compared to the control. Red and red/blue light had a significant effect on increasing CO 2 assimilation at salinity stress. In alkalinity stress, red light had a significant effect on the CO 2 assimilation parameter, and other light treatments had no significant effect on CO 2 assimilation. Parameters of stomatal conductance (g s ) and transpiration rate (E) were also affected significantly by stress and different light spectra, and they decreased under stress conditions compared to the control. In salinity and alkalinity stress, red light had the most significant effect on  increasing of these parameters compared to ambient light treatment. Under non-stress conditions, red and white/yellow light had a significant effect on g s . Also, in these conditions, red and blue light had a significant effect on E.
The internal CO 2 concentration (C i ) under stress conditions was significantly affected by applying different light spectra. Under both stress conditions, blue/red light had a significant effect on reducing this parameter. Blue light has also significant effect in plants under salinity stress. On the other hand, plants with white/yellow light treatment had the highest value of this parameter.
Salinity and alkaline stress decreased water use efficiency (WUE i ) compared to the control. Water use efficiency was improved in the treatment with blue/red light in both stress conditions. Red light has also positively effect in plants under alkalinity stress. Salinity and alkaline stress reduced instantaneous carboxylation efficiency (A/C i ) compared to the control. The highest instantaneous carboxylation efficiency in the control and salinity treatment was observed in blue/red light. In alkalinity stress, the highest A/C i was observed in red and blue light treatment.

Prompt chlorophyll a fluorescence
Both, salinity and alkalinity stress significantly decreased the fluorescent transients compared to non-stresses plants in all light treatments, especially at the I and P steps. However, different light spectra reduced the stress effects and increased the fluorescent transients compared to the treatment without supplementary light. Under salt stress, the higher course in all points has the curve in plants with addition of blue light. In I and P points also addition of red light caused positively changes. The positively changes under alkalinity stress were only in I and P points and they were caused by red, blue/red and white/yellow light (Fig 4).

Transients of chlorophyll fluorescent and calculated curves
The relative variable fluorescence curve was created to explore the effects of stress and light spectrum interaction on transient dynamics, For a detailed evaluation of stress conditions and changes in light spectra in OJIP fluorescence kinetics, we provide differential curves for the L and K bands that occur during the transient O to J. The curves of these bands were calculated by subtracting the amount of normal fluorescence (between O and K, O and J, respectively) recorded in the control plants from that recorded in the plants under stress and light spectra (Figs 5 and 7).
Stress conditions and different light spectra had a significant effect on ΔW L and ΔW K parameters. Salinity and alkalinity stress increased the L and K bands compared to the control treatment (Figs 5 and 7). The blue, blue/red and white/yellow light caused the largest increase in the K-band in plant treated by salt, and white/yellow light caused the largest increase in the L-band compared to other light spectra. In plants under alkalinity stress both, K-band and Lband decreased in all light treatments.

JIP-test parameters
OJIP transients were converted into biophysical parameters: specific energy fluxes, performance indexes, quantum yield for primary photochemistry, and slopes and integrals [8]. The basic parameters derived from the extracted data were next normalized, which made it possible to compare the values measured in plants treated with LED light to plants treated with ambient light only. Salinity and alkalinity stress and different light spectra caused significant changes in these parameters compared to the control treatment (Fig 8 and S1 Table). Blue/red, blue and red light influenced positively on PI ABS and PI total parameters in plants under salt stress. Light with these spectra has also positively influence on F v and F v /F o parameters. Blue, white/yellow red light influenced positively the majority of parameters in plants under alkalinity stress: Area, F m , F v , F v /F o , φ Po , φ Eo , C Eo, PI ABS and PI total . Moreover, Red light influenced φ (Ro) parameter.
Analysis of ANOVA (Table 5) shows that different light spectra, stresses, and interactions affect the JIP test parameters. The stress treatments induced a decrease in F m and increased F O and, as a result, a decrease in F V . The use of light spectra increased F m and F v . The performance indexes (PI ABS and PI total ) were significantly affected by treatments. Both stress treatments, especially alkalinity stress, caused a significant decrease in these parameters, and the use of light spectra, especially red light, increased these parameters compared to the control. Both stress treatments significantly reduced Quantum yield parameters (φ Po , φ Eo , φ Ro , C Eo , δ Ro ), and showed that these parameters are sensitive to environmental stresses. Different light spectra reduced the effects of stress and increased Quantum yield parameters compared to the control, and red light had the highest impact. Both stress treatments affected reaction centers, increased the ABS/RC, DI o /RC, and TR o /RC parameters. We observed that alkaline stress significantly increases the effective antenna size at each reaction center (ABS/RC). ET o /RC and especially RE o /RC decreased under salinity and alkalinity stress treatments, and red light had the most significant effect on these two parameters and increased them. The V J , dVG/dt o , and dV/dt o parameters increased in plants treated by salt and NaHCO 3 , and red light reduced stress on V J and dV/dt o parameter more than other light spectra.

Discussion
The possibility of using LEDs as a light source is a major issue in terms of improving plant efficiency. The life cycle of plants is not only influenced by the intensity of light, but also by the composition of light spectrums. Therefore, in this work, we compared four different light spectra in terms of photosynthesis performance and chlorophyll a fluorescence parameters of strawberry cv. Camarosa under salinity and alkalinity stress conditions. Numerous studies have shown that salt and alkaline stresses inhibit photosynthesis in different plant species [29,30]. There is also demonstrated, that these stresses have a large effect on PSII [31,32]. They affect photosynthetic parameters, which indicates that the photosynthetic apparatus is significantly sensitive to stresses [33]. However, there is a lack of knowledge, whether and how growth under different light spectrums can protect the PSII from salt and alkalinity stresses.
Our studies confirmed that salt stress affects CO 2 assimilation. Moreover, we proved that both stresses effects on chlorophyll fluorescence. The stress of salinity and alkalinity affected both the parameters of F o and F m . An increase in F o and a decrease in F m indicate a block in the transport of electrons from P680 to Q A and the development of non-radiative dissipation of the excited states of PSII antennae chlorophyll [34]. A significant decrease in F m indicates inhibition of electron flow in PSII and could be due to non-photochemical quenching, degradation of the D1 protein, or inactivation of RC PSII [11]. Kalaji et al., reported similar findings from the effect of salt stress on chlorophyll fluorescence [35]. A decrease in the maximum quantum yield of PSII (φ Po ) shows that the stress conditions inhibit the redox reaction after Q A and slow the electron transport between Q A and Q B . A lower δ Ro level indicates a decrease in electron outflow due to the deactivation of ferredoxin NADP + -reductase in PSI [36]. Under salinity stress, PSII activity decreases due to harmful effects of salinity on manganese clusters, and also due to the separation of plastocyanin and cytochrome c553, PSI activity decreases [37]. In salt-stressed wheat leaves, PI ABS decreased due to both ionic and osmotic stress [9], and the decline of PI ABS was associated with a decrease in (F m -F J )/F v [38]. It has been reported that in alkaline conditions, Sm and PI index in strawberry plants has decreased [18]. Deng et al. reported similar results under salinity and alkalinity stress. The flux ratios ABS/RC, TR o /RC, and DI o /RC increased due to salinity and alkalinity stresses [39]. ABS/RC (total number of photons absorbed by Chl molecules) is obtained by dividing the total number of RCs by the total number of active RCs. As the number of inactive reaction centers increases, so does the ABS/RC ratio [40], which decreases the transport of electrons in active RC (ET o /RC) and reduces the final acceptor in PSI (RE o /RC). In plants grown in red, blue, and white/yellow LED light, electron transport flux per reaction center and the possibility that the trapped exciton will transfer the electron in the transport chain beyond the Q A -(ψo) increased (Fig 6). The results showed that plants grown in red, blue, and white/yellow LED light could bring more electrons into the electron transport chain and beyond Q A from absorbed photons, and this indicates that these plants after exposure to stress, positively regulate energy levels in the reaction centers [41]. According to our studies, changes in light spectra significantly affect the photosynthetic system of strawberry plants. These conclusions were in line with the results by Hogewoning et al., or Macedo et al., [42,43]. We also found that under salinity stress, red and red/blue light had a positive effect on net-photosynthesis rate. In alkalinity stress, only red light partially mitigated the decrease in this parameter. Salinity and alkaline stress decreased water use efficiency (WUE i ) compared to the control, but blue/red light had a significant positive effect on this parameter and maintain it at the control level under salinity. WUEi under salt stress was also improved by red and blue light used separately. In alkalinity stress, the highest A/C i was observed in red and blue light treatment, and the other treatments had no significantly different from each other. The red and white/yellow light had the most significant effect on increasing fluorescence in plants under both stresses. Plants absorb mostly blue and red light (about 90%). Besides of absorption by photosystems blue light can indirectly affect stomatal opening which can be independent on photosynthetic activity (CO 2 decrease in the leaf) and thus blue light can increase transpiration without significant effect on photosynthesis [44]. The opening of the stomatal with red light is due to the response of the stomatal guard cells to a decrease in the intercellular concentration of CO 2 and the direct reaction of the chloroplast guard cells to red light [45]. Blue light is also essential for chlorophyll biosynthesis, and red light is also vital in this process [42]. The blue and red light combination is used in commercial research and horticulture because of the vital role of these wavelengths in photosynthesis. The lack of one of them (red or blue light) decreases the efficiency of photosynthesis. However, when the LED light is used as a complementary light in greenhouse lighting conditions, they may have different effects, and some wavelengths may be more effective.
The stomatal closure is the first defense of the plant against salinity and alkalinity stresses. Salinity affects photosynthesis by stomatal closure, reducing carbon uptake, and damaging photochemical reactions [46]. In stress conditions, and due to increased penetration resistance in stomatal and mesophilic cells, CO 2 availability is affected [47], which reduces electron transfer to the final acceptor.
There is a high correlation between an increase in CO 2 assimilation rate and an increase in PI ABS , PI total, and quantum yield values, which provides evidence for changes in OJIP fluorescence rise kinetics using different light wavelengths under stress conditions with changes in the total capacity of photosynthesis. Under these conditions, the process of photosynthesis is regulated to maintain a balance between electron transfer reactions and carbon regeneration metabolism [48].
All the processes described above may point the very complex relationship between wavelength, environmental stresses and photosynthetic response. Additionally, studies on optimization of greenhouse light sources clearly pointed that the optimum light spectrum depends on plant species and developmental stage [49].
Our results indicate that another factor which can be considered in light optimization are potential abiotic stresses. The choose of optimum light conditions under stress may optimization of photosynthetic activity it is possible to balance the light phase energy production and the demand for this energy for carboxylation by the mentioned developmental effects and stomatal conductance. When the stress decreased energy demands the energy surplus in photosystems leads to damages or to reduction of long-term light phase capacity [50]. By changing light wavelengths and intensity we can regulate the energy supply to adjust them to the demand and also by manipulation of stomatal closure we can affect the demand and additionally water use, which may be also important for more sustainable agricultural production.

Conclusions
The results of the present work suggest that the use of different light spectra can partially mitigate the deleterious effects of salinity and alkaline stress on photosynthesis of strawberry plants. Red and blue/red light had a significant effect on enhancing CO 2 uptake under salinity stress conditions while under alkaline stress, only red light had a significant effect on its magnitude. We found that some chlorophyll fluorescence parameters can be used as bio-indicators to optimize the light spectrum for production systems that require a more resilient response to non-optimal growth substrate (too alkaline peat-free growth media) or nutrition (organic systems). Analysis of chlorophyll fluorescence parameters have demonstrated the changes in   Supporting information S1